Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

Abstract

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis ( Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.

Author Summary

Currently, approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis ( Mtb), the bacterium that causes tuberculosis (TB). A central question in TB research is to identify the mechanisms that allow the organism to persist for long periods of time in humans. The mycobacterial cell wall has a high lipid content and contains several important lipid groups, including poly- and di-acyltrehaloses (PAT/DAT), sulfolipids (SL-1), and phthiocerol dimycocerosates (PDIM). These lipids are produced and actively secreted during infection to subvert the host immune system, eventually leading to Mtb persistence. We have discovered that the regulatory protein WhiB3 controls the differential production of PAT, DAT, SL-1, and PDIM and the storage lipid triacylglycerol (TAG) in response to fluctuations in the intracellular redox environment. We demonstrated that WhiB3 directly regulates lipid production by binding to the promoter regions of lipid biosynthetic genes in a redox-dependent manner. We also discovered that through this regulatory process, WhiB3 controls fatty acid metabolism and maintains intracellular redox homeostasis by channeling toxic reducing equivalents into lipid anabolism. Thus, our results suggest that Mtb lipid anabolism functions as a reductant sink to neutralize the reductive stress associated with the catabolism of host lipids during infection. These findings may serve as a model foundation for how pathogens adjust their metabolism to cope with stresses encountered during infection.